1. Field of the Invention
The present invention relates to a protein of a G-protein coupled receptor having an eicosanoid as a ligand (more specifically, 5-oxo-6E, 8Z,11Z,14Z-eicosatetraenoic acid, hereinafter referred to as 5-oxo-ETE), or a gene thereof. The present invention also relates to a process for screening a candidate for a pharmaceutical compound, using the same. Further, the present invention relates to a pharmaceutical composition comprising an antagonist of the receptor, and to a method for treatment, etc. using the antagonist.
2. Background Art
Physiologically active substances such as many neurotransmitters, hormones, autacoids, etc. regulate biological functions through interactions with specific receptors which exist on the cell surface. Most of these receptors are coupled with a GTP-binding protein (hereinafter referred to as G-protein), which is a trimeric protein existing in a cell, and it has been known that signals are transduced intracellularly via an activation of the G-protein. These receptors therefore are generally referred to as G-protein coupled receptors. Any of the G-protein coupled receptors are known to share a common structure including 7 transmembrane domains.
The G-protein is a trimeric protein consisting of α, β and γ subunits, and in a ground state, it exists as an inactivated form (GDP-binding form) in which these subunits are associated. By the G-protein coupled receptor which has been stimulated by a ligand, the inactivated form (GDP-binding form) of the G-protein is converted to an activated form (GTP-binding form), and it is dissociated into an a subunit (Gα) which is bound to GTP, and a β/γ subunit complex (Gβγ). Then, the GTP-binding α subunit (Gα) (or occasionally Gβγ) controls effectors such as adenlylate cyclase, phospholipase C, etc., to transduce signals.
Further, the G-protein, especially Gα, has a variety and it is known that different kinds of effectors are regulated depending on the kinds of Gα. In general, the G-protein coupled receptor activates a specific type of G-protein, through which a specific type of signal transduction is made intracellularly.
As the G-protein coupled receptor, there have been known α- and β-adrenaline receptors, muscarinic acetylcholine receptors, adenosine receptors, angiotensin receptors, endothelin receptors, gonadotropin-releasing factor receptors, H1- and H2-histamine receptors, dopamine receptors, metabotropic glutamate receptors, somatostatin receptors, etc.
Each of the above receptors plays an important roll in vivo, as a target of physiologically active substances. Moreover, it is a significant fact that many of the medicaments which have been known to date are ligands, or agonists and antagonists whose targets are the G-protein coupled receptors.
From these facts, the G-protein coupled receptors have been drawing attention as a target of research and development of the pharmaceuticals. It has been earnestly desired to find out a novel G-protein coupled receptor, to identify a ligand thereof, and to find out a method for screening or identifying the agonists and antagonists thereof, since they lead to screening of a novel candidate for pharmaceutical compound.
With respect to the eicosanoid receptors, the followings have been known. Those generally referred to as eicosanoids include prostaglandin, prostacyclin, thromboxanthine, leukotriene, and an eicosatetraenoic acid such as 5-oxo-ETE, 5-hydroxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (5-HETE), etc. As the receptors having eicosanoid as a ligand, there has been reported prostaglandin EP1, EP2, EP3, EP4, F2α receptor, prostacyclin PI2 receptor, thromboxanthine TA2 receptor, leukotriene B4 receptor, which are all G-protein coupled receptors (Narumiya, et al., Physiol. Rev., Vol. 79, pp. 1193-1226, 1999; Mohammed Akbar, et al., J. Biol. Chem., vol. 271, pp. 18363-18367, 1996). However, a receptor having an eicosatetraenoic acid such as 5-oxo-ETE and eicosatrienoic acid as a ligand which has been identified in the present invention has not been reported to date.
Further, with respect to the 5-oxo-ETE, (5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid), the followings have been known.
5-Oxo-ETE is a substance derived from a fatty acid having 20 carbon atoms, such as an eicosanoic acid, and it is synthesized from arachidonic acid which is a biosynthetic intermediate of prostaglandin and leukotriene, through 5-HETE (see FIG. 1 below).
It has been known that 5-oxo-ETE induces eosinophil and neutrophil migration (Schwenk, et al., J. Biol. Chem., Vol. 270, pp. 15029-15036, 1995; Guilbert, et al., Am. J. Respir. Cell Mol. Biol., Vol. 21, pp. 97-104, 1999; Stamatiou, et al., J. Clin. Invest., Vol. 102, pp. 2165-2172, 1998).
It has been known that 5-oxo-ETE is a growth factor and a maintenance factor of prostate cancer, breast cancer, lung cancer, pancreas cancer, and mesothelioma, and inhibition of the synthesis of the same will lead to apoptosis (Ghosh, et al., Biochem. Biophys. Res. Commun., Vol. 235, pp. 418-423,1997; Ghosh, et al., Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 13182-13187, 1998; Avis, et al., FASEB J., Vol. 15, pp. 2007-2009, 2001; Avis et al., J. Clin. Invest., Vol. 97, pp. 806-813, 1996; Ding, et al., Biochem. Biophys. Res. Commun., Vol. 261, pp. 218-223, 1999; Romano, et al., FASEB J. Vol. 15, pp. 2326-2336, 2001).
An object of the present invention is to provide a G-protein coupled receptor having eicosanoid as a ligand, and a gene thereof.
Further, an object of the present invention is to provide a method for screening, identifying, and characterizing a ligand and an effector (an agonist or an antagonist) for the receptor protein.
Further, an object of the present invention is to provide a novel method for treatment, etc., using an antagonist of the receptor (the G-protein coupled receptor having an eicosanoid such as 5-oxo-ETE as a ligand).
The present inventors have isolated a full length human cDNA encoding a novel G-protein coupled receptor (hereinafter also referred to as TG1019). Moreover, they have successfully expressed the receptor protein in the cell, using recombinant DNA technology. Further, they have identified a ligand for the receptor, and have found out that the receptor has an eicosanoid (5-oxo-ETE, etc.) as a ligand (Hosoi, et al., J. Biol. Chem., Vol. 277, pp. 31459-31465, 2002).
In addition, the present inventors have found out that migration of eosinophils and neutrophils by 5-oxo ETE are induced through this receptor. Yet further, they have found that an antagonist of the receptor induces apoptosis in cancer cells, to accomplish the present invention.